This project will focus on gaps in knowledge that must be overcome to advance precision, genotype-specific intervention for cystic fibrosis (CF). First, we will characterize disease-associated alleles, many of which are extremely rare, and have been assigned to an incorrect or incomplete mechanistic category. Other variants represent novel defects not evaluated previously. Our goal will be to elucidate limitations of the conventional diagnostic categories for disease-associated CF alleles, and to address modulator drug responsiveness (i.e., ?theratype?) for a number of rare CFTR mutations that have not been previously tested in this manner. We will also provide examples of ways the recently solved CFTR cryo-EM structure can help define disease- associated mutations and their effects on CFTR folding (Aim 1). Our studies will provide cell systems for molecular phenotyping of alleles representing thousands of patients, and should be of value to the scientific community, clinicians, and patients with the disease. Second, new model systems are needed that predict in vivo benefit using drugs such as ivacaftor or Orkambi (which contains both ivacaftor and lumacaftor). As a test of emerging cell systems for this purpose, we will generate iPS lines corresponding to 20 of the CFTR variants profiled under Aim 1. Experiments such as these will provide a first evaluation of: 1) the extent to which iPS cells can be used effectively as polarized monolayers according to the best available and innovative protocols developed by our laboratory and others, 2) whether these model systems exhibit detectable modulator response in vitro, 3) whether iPS cells are similar in molecular phenotype to widely used (FRT) cell models encoding the same mutations for this purpose, 4) usefulness of leading edge gene editing technologies in iPS cells to produce first-line systems, and 5) practical features of iPS technology vis--vis personalized therapeutics, including the extent to which an individual patient sample can be expanded effectively, durability of CFTR expression, function in vitro, stability of monolayers developed in this manner (Aim 2), etc. Finally, we will take up the challenge to establish the practical usefulness of our work by facilitating a new intervention in CF patients carrying the P67L/F508del genotype, a case study emblematic of hundreds of other CFTR variants. We have a compelling body of data indicating that P67L patients will benefit significantly from a drug such as Orkambi. Aim 3 will formally pursue this assertion in a clinical setting by directly testing human subjects with this very rare allele. If successful as predicted, a group of patients who currently have no effective modulator therapy will directly benefit. Moreover, because we believe it is likely that iPS cells will recapitulate clinical efficacy, the data is intended to help establish an in vitro surrogate that will improve drug access and precision therapeutics for a population of CF patients carrying rare variants other than P67L.